This invention concerns an improvement in the art of the transplantation of tissue for medical purposes. Specifically it concerns methods, vectors and compounds useful in preventing the expression of certain transplantation antigens on the surface of cells to be transplanted. More specifically it concerns the blockage of the cell surface expression of one particular class of proteins the genes for which are located in the major histocompatibility complex (MHC).
In cases of extreme and life threatening conditions such as renal, liver and cardiac failure, physicians have successfully transplanted tissues between genetically distinct individuals. Even when care is taken to reduce the genetic differences between the host and donor, the recipients of these grafts must usually be given drugs that reduce the activity of their immune system so that the graft is not rejected. Such immuno-suppression entails substantial risks. Even when such immuno-suppression is well tolerated, there are considerable difficulties attendant in minimizing the antigenic differences (matching) between the donor and the recipient that increases the costs and reduces the availability of this mode of therapy. Furthermore, not all tissues can be successfully transplanted between genetically distinct persons.
In principle, for example, diabetes mellitus, one of the most common and, in the longterm, one of the most debilitating chronic diseases, could be xe2x80x9ccuredxe2x80x9d by a successful transplant of the tissue that secrete insulin, the islets of Langerhans. Despite the magnitude of the problem, the availability of islets from cadaveric donors and the successful experience in other situations, e.g., renal, cardiac and hepatic transplantation, there is presently no practical protocol that routinely provides for the survival of histoincompatible islet cells.
The probable causes for this absence may be several. In diabetes mellitus, the disease itself makes immuno-suppression especially hazardous, as diabetics are highly susceptible to infection. Also it appears that some immuno-suppressive agents, e.g., cyclosporine, are directly toxic to islets in high doses and can adversely affect graft survival, while others, e.g., glucocorticoids, are known to increase the subject""s insulin requirements and may indirectly jeopardize both the survival and the beneficial effects of the graft. Lastly, in many instances diabetes results from autoimmunity directed toward the islets. To the extent such individuals are immunologically xe2x80x9cprimedxe2x80x9d towards non-allelic islet antigens, their graft rejection would be accelerated. Thus, as an alternative to the present methods of promoting graft survival, which reduce the host""s immunity, there is a need in the transplantation art for a method to specifically render the graft invisible to the host""s immune system or resistant to its effects.
2.1 The Mechanism of Graft Rejection
Except between genetically identical individuals, e.g., identical. twins, the cells of each person are recognized by the immune system of others as foreign, i.e., the immune system responds to the graft just as it would respond to a parasite or virus. This so called allograft reaction, unless treated by immune-suppression, leads to the rejection and loss of the graft. The allograft reaction is caused by the recognition of histocompatibility antigens on the surface of the cells of the graft by the lymphocytes of the recipient individual. With rare exceptions, e.g., the ABO blood groups in transfusions, the host lymphocytes of greatest importance to an allograft reaction are of the type that recognize antigen directly on cell surfaces, so-called thyme lymphocytes (T-lymphocytes).
From the fact that only grafts between genetically identical individuals are truly stable, one may conclude that. almost any protein can become a histocompatibility antigen when the host""s and recipient""s genes encoding it are different. This surmise has been experimentally verified. However, the rapidity with which an allograft is rejected varies greatly depending upon the nature of the histoincompatibility between the host and graft. In particular, every species of higher vertebrates contains a closely linked complex of multiple genes at which genetic differences are found to cause the most rapid and severe allograft reactions. This complex of genes is called the Major Histocompatibility Complex (MHC) of the species. Several types of proteins are encoded within the MHC, including two classes of cell surface proteins, conventionally called class I and class II MHC products. In humans the class I products are also referred to as HLA-A, HLA-B and. HLA-C, and the class II products are termed HLA-DR. Many cell types, such as xcex2-cells, express only MHC class 1 products.
Class I and class II MHC products are not only histocompatibility antigens, they are also central to antigen recognition by T-lymphocytes. Although the different T-lymphocytes from an individual can recognize and distinguish among perhaps millions of potential antigens, each of that individuals T-lymphocytes can recognize its particular antigen only when the antigen is either an MHC class I or class II product or is physically complexed with one. These observations suggest that the life of an allograft could be extended indefinitely were some mechanism available to prevent the expression of MHC products altogether by the graft.
2.2 The Viral Products That overcome Host Immunity
The problem of avoiding a host""s immune reaction is of interest not only to transplant surgeons but also to parasites such as viruses. Viruses, of course, are able to reproduce only within a viable cell of the infected host. Viruses frequently kill the infected cell when they redirect the cellular machinery towards the production of viral particles (virions). Surprisingly then, one of the major host defenses against viruses are cytotoxic T-lymphocytes (CTL) which kill the infected cells. The host apparently prevents, to some significant degree, the further release of virions by killing the infected cell when viral antigens can be detected. In response, certain viruses have evolved mechanisms to reduce or avoid the host""s immune attack on the host""s cells in which the virus is replicating (reviewed, Gooding, L. R., 1992, CELL 71:5-7) by blocking the presentation of newly synthesized antigens. Some of these mechanisms apparently specifically prevent the maturation of MHC class I products onto the cell surface. Because the complex of MHC product and antigen is formed as the product matures, the display of newly synthesized viral antigens is blocked.
Researchers have recognized that adenovirus infected cells are poorly recognized and lysed by CTL. To determine which viral products are particularly involved, the isolated E3 region of adenovirus was co-transfected, along with a selectable marker, into the 293 embryonic kidney cell line, which had been transformed with adenovirus and contained adenovirus early genes products, that rendered the adenovirus E3 promoter active.
Stable transfectants were isolated by selection of the marker gene and a single clone expressing high levels of gp19 was selected for further study. This transfected clone was found to have undergone an about 5-10 fold reduction in the amount of HLA-A and somewhat smaller reduction in total MHC class 1 products. There were no studies of the functional consequences of this reduction. Burgert, H-G and Kvist, S., 1985, CELL 41:987-97.
Subsequently, these authors introduced a murine MHC class I product (Kd) into the selected clone and showed that this clone was resistant to Kd-specific CTL, Burgert, H-G, 1987, PROC.NATL.ACAD.SCI. 84:1356-60. Studies of murine immune responses to adenovirus antigens, using varying deletion mutants spanning the E3 region and anti-MHC monoclonal antibodies, establish that the gp19 protein (gp19) both binds murine MHC product and is necessary and sufficient for the inhibition of its expression on the surface of the embryonic kidney cell. Burgert, H-G., and Kvist, S., 1987, EMBO J. 6:2019-26; Rawle, F. G. et al., 1989, J.IMUUHOL. 143:2031; Cox J. H, et al., 1991, J.EXP.MED. 174:1629-37; Hermiston, T. W. et al., 1993, J. VIROLOGY 67:5289.
The issue whether the inhibition by gp19 of the expression of MHC class I products is a general effect, or one specific to adenovirus transformed cell lines remains incompletely resolved. The expression of gp19 in cells other than the embryonic kidney line of Burgert is associated with the inhibition of glycosylation of MHC class 1 heavy chain indicating that this molecule does not exit the endoplasmic reticulum, Burgert, 1985, supra.; these or other processes block the presention of viral antigens in acutely infected cells, Cox, J. H., et al., 1990, SCIENCE 247:715-718. However, there was no evidence of a reduction in the total amount of cell surface MHC class I product other than in the adenovirus-transformed cell lines studied by Burgert. Routes, J. M., and Cook, J. L., 1990, J.IMMUNOL. 144:2763.
The scientific literature does not establish whether the chronic presence of gp19, in quantities attainable by present methods without cytotoxic effects, will render a cell resistant to CTL. CTL are fully active against targets that have between 0.1%-0.2% of the normal concentration of MHC class I products. Vitiello, A., et al., 1990, SCIENCE 250:1423. Thus, the relative reduction in MHC class I expression needed to prevent CTL activity is approximately 100 fold greater than that observed in the most strongly inhibited example of Burgert. Thus, while the evidence is strong that gp19 causes an acute failure of adenovirus infected cells to present newly synthesized antigens and blocks the cell-surface-display of newly synthesized MHC molecules, it does not indicate whether gp19 will be sufficient to permanently eliminate a sufficiently large fraction of MHC. class I products from the surface of cells expressing gp19 to render the cells resistant to the effects of CTL.
Other proteins that can be used to inhibit the expression of newly synthesized MHC class I products can be found. A herpes simplex virus (HSV) virus protein, ICP-47, having similar properties to adenovirus gp19 has been described. York, I. A., et al., 1994, CELL 77:525-35. Human cytomegalovirus appears also to express a protein having this activity. Beersma, M. F. C., et al., 1993, J. IMMUNOL. 151:4455-64.
2.3 Treatment of Diabetes Mellitus by Transplantation of B-cells
Diabetes mellitus is a disease of glucose regulation characterized by excessive blood glucose caused by either an absolute deficiency of insulin-secreting xcex2-cells or, more commonly, by a combination of hyporesponsive xcex2-cells and the resistance of the subject to the effects of insulin. Although there is extensive clinical evidence that insulin therapy combined with strict adherence to dietary control, can control the acute effects of diabetes such as ketoacidosis, such therapy does not stabilize the blood glucose levels sufficiently to prevent the long term ophthalmologic, neuropathic, nephropathic and arterial complications of the disease.
To prevent these complications, physicians have experimented with the transplantation of islets obtained from the pancreas of human cadaveric donors. These reports demonstrate the practicability of techniques to obtain and isolate islets from cadaveric donors and to implant the islets into the liver of diabetic subjects by injection of isolated whole islets into the portal circulation. Scharp, D. W., et al., 1990, DIABETES 39:515-8. The results of a series of longitudinal studies of several patients, however, show that, at levels of immuno-suppression appropriate for renal or hepatic allografts, islet cell transplants do not routinely survive and function well enough to eliminate the need for exogenous insulin. Ricordi, C., 1992, TRANSPLANTATION 53:407-414. Among the explanations available for this phenomenon is that many diabetic patients have developed an auto-immunity to islet cells, a so-called insulinitis, which may cause an unusually intense allograft reaction to the transplanted islets. Alternatively, the cyclosporine appears to have a direct- toxicity for xcex2-cells and glucocorticoids have known diabetogenic effects that antagonize insulin and jeopardize graft function and survival. Whatever the mechanism, the results of these studies show that conventional immuno-suppression is generally not successful in enabling islet transplantation. Thus, there is an unmet need in medical practice, for a method to transplant islet cells between histoincompatible individuals.
There have been attempts to answer the question whether the complete suppression of MHC class I products from the surface of the xcex2-cells would allow indefinite survival of allograft islets without immuno-suppression, in murine experimental systems. These studies employ transgenic animals lacking both maternal and paternal copies of the xcex22-microglobulin gene, the gene necessary for the expression of functional MHC class 1 products. These studies show that islet grafts of normal recipients from xcex22-microglobulin deficient donors, frequently, though not invariably, survive for an indefinite period. Orsorio, R. W., et al., 1993, DIABETES 42:1520-27; Markman, J. F., et al., 1992, TRANSPLANTATION 54:1085-89. However, when such islets were transplanted into NOD mice, which spontaneously develop an autoimmune diabetes and are considered to be a model for human type 1 diabetes, there was no prolongation of the survival of the grafted islets. Markman, supra. The failure of xcex22-microglobulin deficient islets to survive in NOD mice does not imply that MHC-class 1 restricted CTL are not essential to the development of diabetes in the NOD model. Experiments show that xcex22-microglobulin deficient-NOD mice, which show markedly reduced levels of MHC class I products in all tissues do not develop either diabetes or the lymphocytic infiltrate of their islets, a condition which is called insulinitis. Serreze, D. V., et al., March 1994, DIABETES 43:505-9; Wicker, L. S., et al., March 1994, DIABETES 43:500-4. Together these results establish that, at least in mice, the removal of MHC class I products from the surface of islets by means of deletion of the xcex22-microglobulin gene of the islets does not lead to a successful therapy in a mouse having a CTL dependent auto-immune diabetes.
To meet the need for an effective method to prevent the rejection of islet grafts, some have proposed the use of xe2x80x9cmaskingxe2x80x9d agents, such as F(abxe2x80x2)2 anti-HLA antibodies to prevent recognition by the CTL of the MHC class I product. Data indicate that the pre-treatment of human islets with anti-HLA antibody effectively prolongs the survival of transplants into the renal-subcapsular space of murine hosts to as much as 200 days; while absent such pretreatment rejection occurs within 7 days. U.S. Pat. No. 5,283,058 to D. Faustman. The examples presented by Faustman concern xenografts, particularly the engraftment of mice with human islets. No data are presented by Faustman showing that this method would be effective in preventing allo-rejection, i.e., the rejection of murine islets by mice. Because xenograft rejection is most often of the hyperacute type mediated by preformed antibodies, the projection of the reported results concerning the Faustman technique to the case of allo-transplantation is of uncertain outcome. Faustman also suggests that unspecified viral proteins may be used in conjunction with, or as an alternative to, the use of masking agents to decrease MHC class I expression to prolong islet graft survival.
The invention provides a method and vectors to express a gene, derived from a virus, that blocks the intracellular transport and/or intracellular maturation within the graft of proteins called MHC class I products. Without limitation as to theory, it is believed that blocking the appearance of this class of proteins on the transplanted cell""s surface, prevents the host""s immune system from rejecting the graft. In one embodiment, the invention is directed towards engrafting the cells that secrete insulin, which are called alternatively, pancreatic xcex2-cells and islet cells, and thereby provide a treatment of diabetes mellitus.
The present invention provides a method of rendering cells more readily transplantable between histoincompatible individuals; the pancreatic islet is a non-limiting example of one such type of graft. The method comprises obtaining a population of cells to be transplanted, e.g., by explantation from a cadaveric donor, and introducing into substantially all of the cells of the population a vector that makes a nucleic acid product.
In one embodiment the vector acts by producing a protein expression product that prevents the expression of the MHC class I products. In another embodiment, the vector may act by producing protein products which inhibit. a variety of host immune responses important in graft rejection. In an alternative embodiment the vector may act by producing a nucleic acid expression product having a specific nucleic acid that interferes with the production of the MHC class I products.
Any population of cells suitable for beneficial implantation into a subject may be employed. Suitable cells are preferably stable and non-transformed. Stable, non-transformed cells are cells having a life-span that is at least several weeks and most. preferably many months or years and are cells with normal growth characteristics such that upon transplantation into an immunodeficient host, such as a nude mouse, there is no tumor formed.
Proteins suitable for the practice of the invention can: be found among human virus proteins; adenovirus (Ad) gp19 and HSV ICP-47 are representative non-limiting examples. The invention further provides for vectors to express MHC class I inhibitory proteins in the population to be transplanted. Expression vectors suited for the present invention necessarily must be able to successfully transfect substantially all the cells, which is preferably more than half of the cells, more preferably greater than 80% of the cells, and most preferably greater than. 95% of the cells. Such vectors must not produce other cytopathic effects that would interfere with other normal. functions of the cells to be transplanted. Suitable examples of such vectors include replication defective virus vectors based on HSV and adenovirus,. and vectors based on the satellite virus, adeno-associated virus (AAV). The invention further provides populations of non-transformed cells, i.e., cells whose growth is controllable, that lack detectable quantities of MHC class I products on their surface as determined by their ability to be targets of lysis by T-lymphocytes of the appropriate specificity.